Specific-mutational patterns of p53 gene in bladder transitional cell carcinoma among a group of Iraqi patients exposed to war environmental hazards.

Al-Kashwan et al. BMC Research Notes 2012, 5:466
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Research Notes

RESEARCH ARTICLE Open Access

Specific-mutational patterns of p53 gene in
bladder transitional cell carcinoma among a
group of Iraqi patients exposed to war
environmental hazards

Thekra A Al-Kashwan 1 , Massoud Houshmand 2 , Asaad Al-Janabi 3 , Alice K Melconian 4 , Dhafir Al-Abbasi 3 ,
Muhammad N Al-Musawi 5 , Maryam Rostami 2 and Akeel A Yasseen 3,6 *

Abstract

Background: To unfold specific-mutational patterns in TP53 gene due to exposures to war environmental hazards
and to detect the association of TP53 gene alteration with the depth of bladder cancer.

Methods: Twenty-nine bladder carcinomas were analyzed for TP53 alterations. PCR-single strand conformational
polymorphism analysis, DNA sequencing and immunohistochemical analysis using monoclonal mouse anti-human
p53 antibody (Clone DO-7) were employed.

Results: TP53 gene mutations occurred in 37.9% of the cases while TP53 overexpression occurred in 58.6%. Both of
them were associated with deep invasive-tumors. Single mutations were seen in 63.6%, whereas only 27.3% have
shown double mutations. Four mutations were frameshifted (30.8%); two of them showed insertion A after codon
244. There was no significant association between TP53 mutations and protein overexpression (P>0.05), while a
significant association was observed between TP53 alterations and tumors progression (P < 0.01).

Conclusion: The infrequent TP53mutations, especially insertion A and 196 hotspot codon, may represent the
specific-mutational patterns in bladder carcinoma among the Iraqi patients who were exposed to war
environmental hazards. TP53 alteration associated with bladder cancer progression should be analyzed by both
mutational and protein expression analysis.

Keywords: Bladder cancer, TP53 alteration, Specific mutation, Immunohistochemistry

Background

Bladder cancer (BC) is the most common malignancy
affecting urinary system comprising the seventh most
common cancer worldwide with male predominance
[1,2]. In Iraq, the incidence of bladder cancer is the
fourth most common type of cancer (in men and eighth
most common in women) [3]. The most common type
of bladder cancer is transitional cell carcinoma (TCC)
accounting for more than 90% of the cases [2]. It repre-
sents one of the first tumors that have been associated

* Correspondence: a.yasseen@hotmail.com

department of Pathology and Forensic Medicine, Faculty of Medicine,
University of Kufa, Kufa, Iraq

department of Pathology and Forensic Medicine, Faculty of Medicine,

University of Kufa, Kufa, P.O. Box 21, Najaf Governorate, Iraq

Full list of author information is available at the end of the article

(3 BioMed Central

with environmental risk factors that produce genetic
alterations [4,5].

TP53 gene "the guardian of genome" is the most fre-
quently mutated tumor suppressor gene identified in
human cancer. TP53 inactivation led to diminished con-
trol cell cycle check points, decreased DNA repair, and
increased genomic instability [6-8] . Furthermore, TP53
inactivation has been identified in tumor progression,
metastasis, and aggressive phenotype of bladder cancer
giving rise to what has been considered as a useful gen-
etic biomarker to predict progression associated with
bad prognosis [9-11]. The frequency and the type of
mutations vary from one tumor type to another, ranging
from 5% to 80% depending on the type, stage and eti-
ology of tumor [12]. Almost all TP53 mutations are

© 2012 Al-Kashwan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.Org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

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point missense mutations leading to a functionally de-
fective protein [13]. Approximately 90% of them are
localized in DNA-binding domains encoded by exons 5-
8. In total, about 40% of them are localized at the "hot-
spot" residues R 175, G 245, R 248, R 249, R 273 and R
282 [14]. Indeed, there are considerable numbers of
studies on TP53 gene being as a target for carcinogens,
with a specific TP53 mutations spectrum, mainly G:
C^A: T transitions at CpG and non-CpG sites in blad-
der cancer [7,15,16]. It is well documented that the
spectrum of TP53mutations in bladder cancer differs
from that of lung cancer, even though cigarette smoking
is probably a contributing cause in over one-third of all
bladder cancer cases [17]. Specifically, G^T transver-
sions at CpG are relatively uncommon (about 8% in
bladder cancer versus 27% in lung cancer) whereas CpG
G: C^A: T transition are as twice as common with re-
gard to mutation patterns (22% for bladder cancer ver-
sus 11% for lung cancer) [7].

Inactivation of tumor suppresser genes can occur ei-
ther primarily through mutations, or without any change
in the structure of the given genes. Thus, tumor sup-
pressor function should be analyzed at the level of the
genes as well as at the level of proteins, and in the con-
text of the pathways in which these genes are involved
[18,19]. The mutated p53 protein has a longer half-life,
as compared with normal p53 protein, which can be
detected by Immunohistochemistry (IHC) as a surrogate
marker for mutation [20]. Immunohistochemical positiv-
ity for TP53protein is in general thought to reflect point
mutations of TP53 gene in tumor, although it is not al-
ways synonymous with TP53 mutation [21]. Accordingly,
using both molecular and protein analyses (PCR-based
genetic technique and immunohistochemistry respect-
ively) for detection of TP53 alterations have rational effi-
cacy for mutation detection rather than each one alone
[20,22,23]. The aims of this research were to determine
if any specific-mutational pattern in TP53gene may play
a possible role in bladder tumorogenesis, which might
be resulted from the exposure to the hazardous pollu-
tion of the wars in Iraq. An analysis of the alteration of
TP53gene using combined PCR-based genetic and IHC
analysis to evaluate its association with bladder cancer
progression was conducted.

Methods

Ethical approval was obtained from the local medical
ethics committee, Faculty of Medicine, University of
Kufa. A written informed consent was received from all
subjects before proceeding any further. The study was
designed and conducted in accordance with the tents of
Declaration of Helsinki.

A total of twenty-nine patients (25 males and 4 females)
with transitional cell carcinoma (TCC), diagnosed by

transurethral resection (TUR-biopsy) at the Department
of Pathology of Kufa School of Medicine Teaching Hos-
pital were subjected to the present study. The patients
were randomly selected from the Middle Euphrates area
and south of Iraq. Both regions were potentially exposed
to environmental pollution during the last two decades of
wars. The patients ages ranged between 35 and 85 years,
with a median age of 69.3 years. Histological examination,
grading [24] and staging [25] were performed by two of us
independently. Three were classified as grade I, two as
grade II, and fifteen classified as grade III. Fourteen cases
were superficial TCC [5 as Ta and 9 as Tl], while 15 cases
were deep invasive TCC (T2). Sixteen of our patients had
a history of cigarette smoking for at least 10 years.

TP53 status

TP53 protein expression

TP53 protein expression was estimated using immuno-
histochemical techniques [26-28]. Five micron thick sec-
tions of formalin-fixed paraffin-embedded tissue (FFPE)
were placed on positively charged slides (Fisher scientific
Co., Pittsburgh, PA). These sections were then deparaffi-
nized and rehydrated. For staining enhancement, the
sections were pre-treated with antigen retrieval solution
(0.01 M, citrate buffer, pH6.0, Dako Cytomation/Den-
mark) in water-bath at 95°C for 40 minutes followed by
staining with a monoclonal mouse anti-human TP53
antibody (Clone DO-7, Ready-to-use, DakoCytomation/
Denmark). The antigen-antibody complex was visualized
using Labeled Streptavidin-Biotin 2 System-Horseradish
Peroxidase staining technique (LAB/LSAB2 System-
HRP, DakoCytomation/Denmark). The sections were
then counterstained with Meyer's haematoxylin.

A breast cancer with high TP53expression detected by
immunohistochemical analysis was used as positive ex-
ternal control. Negative controls were obtained by omis-
sion of the primary antibody and by a breast cancer with
negative TP53 expression detected by immunohisto-
chemical analysis. The non-epithelial cells of samples
(lymphocytes, stromal cells and endothelial cells) were
used as negative internal control.

All slides were reviewed independently by two investi-
gators without previous knowledge of tumor grade and
stage or TP53mutation. TP53 expression (nuclear stain-
ing) was evaluated by counting 100 cells/section in five
randomly chosen high-power fields (40x) by light micro-
scope. The extent of nuclear reactivity was classified in
four categories [9,20]; no nuclear reactivity (-), few fo-
cally positive cells (1 to 10% tumor cells) (+/-), hetero-
geneous nuclear reactivity (10 to 50% tumor cells) (+)
and homogenous intense nuclear reactivity (50 to 100%
tumor cells) (++). The samples which demonstrated at
least 10% nuclear reactivity were considered to be TP53-
positive (have an alteration in TP53) [9,10,19,20].

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TP53 mutation
DNA extraction

High-molecular weight DNA was prepared from the
fresh tumor specimens by phenol-chloroform-isoamyl
alcohol method [20,29]. DNA which was extracted from
whole blood samples of healthy looking individuals was
included as normal control with their matched age and
sex with Wizard ® Genomic DNA purification kit (Pro-
mega Company/USA).

PCR-SSCP and DNA sequencing

The mutational analysis was achieved using PCR-single
strand conformation polymorphism (PCR-SSCP) and
direct DNA sequencing methods for TP53 gene exons
5-8. Each exon 5-8 was amplified by PCR. The primer
sequences used were as follows [15]: (1) exon 5/228 bp,
forward: 5/-TTCAACTCTGTCTCCTTCCT-3/ and re-
verse: 5/-CAGCCCTGTCGTCTCTCCAG-3/; (2) exon
6/159 bp, forward: 5/- GCCTCTGATTCCTCATCGAT-
3/ and reverse:5/-TTAACCCCTCCTCCCAGAGA-3/;
(3) exon 7/157 bp, forwarder- AGGCGCACTGGCCT-
CATCTT-3/ and reverse:5/-TGTGCAGGG TGG
CAAGTGGC-3/; and (4) exon 8/214 bp, forward:5/-
TTCCTTACTGCCTCTTGCTT-3/and reverse: 5/-
AGGCATAACTGCAC CCTTGG-3/. Each PCR reaction
was performed in a final volume of 25 ul containing
100 ng DNA, IX PCR buffer, 1.5 mM MgCL2, 200 uM
each dNTP, 0.1 uM of each upstream and downstream
primer, and 1.5u of Taq polymerase (CinnaGen Com-
pany/Iran). PCR was carried out under the following
conditions: an initial denaturation step (95°C for 5 min-
utes) was followed by 35 cycles consisting of (for exon 5)
denaturation at 95°C for 50 seconds, primer annealing at
55°C for 35 seconds and extension at 72°C for 30 sec-
onds; (for exons 6-7) denaturation at 95°C for 50 sec-
onds, primer annealing at 63°C for 25 seconds and
extension at 72°C for 15 seconds; or was followed by
30 cycles (for exons 8) consisting of denaturation at 95°
C for 50 seconds, primer annealing at 65°C for 25 sec-
onds and extension at 72°C for 15 seconds. The final ex-
tension was continued for 10 minutes at 72°C. The PCR
products were analyzed on 1.5-2% agarose gel to deter-
mine the specific band of each exon product and then
analyzed on 12% polyacrylamide gel to another evalu-
ation of specific band purity to ensure the absence of
any unwanted products (non-specific bands) which may
interfere with SSCP and DNA sequencing analysis.

For non-radioactive SSCP analysis, the SSCP analysis
was carried out according to the method of Liechti-Gallati
et al, (1999) [30], for both of the tumor and the control
samples.

The to controls were analyzed. PCR products were
denaturated at 96°C for 10 min at 3:2 dilution of forma-
mide loading dye (SSCP denaturing solution) containing

95% formamide, 100 mM NaOH, 0.25% bromophenol
blue, 0.25% xylene cyanol and thereafter placed immedi-
ately on ice to prevent re-annealing of the single-
stranded product. The denaturized samples, the controls
and the normal PCR product of controls were loaded
quickly into wells of the 12% polyacrylamide gel and run
at 4-10°C/80 V for overnight. The gel was stained with
Sliver Staining and alterations of bands relative

All the samples that revealed mobility shift in their mi-
gration during SSCP screening mutation analysis were
sent to the direct DNA sequencing in both directions:
forward sequencing 5/^3/and reverse sequencing
3/^5/using the same primer sequences and dideoxy
chain termination method. The DNA sequencing was
achieved by Gen Fanavaran Company/Iran. The DNA
sequencing chromatogram was interpreted using Chro-
magen 2.3 version software that allows comparison of a
newly generated sequence of DNA sample with free
DNA sequence. Viewers (available from National Center
for Biotechnology Information (NCBI) website) were
used as reference sequence of gene of interest for
comparison.

Statistical analysis

The Fishers exact probability test and Odds ratios
(ORs), using contingency tables, were applied to analyze
the data using the program statistical package for the
Social Science (SPSS for windows, version 10.0). The
relationships between the variables were assessed using
non-parametric Fisher s exact probability test. A P-value
<0.05 was considered as statistically significant at a level
of 5%. The strength of the associations between the vari-
ables was measured by calculating Odds ratios (ORs)
and confidence intervals (95% CI). Possible categories
for OR are greater than land less than 1. A value greater
than 1 indicates positive association and a value less
than 1 indicates negative association.

Results

Mutational analysis of the TP53 gene in relation to
clinicopathological features
Characteristic of TP53 gene mutation

The PCR-SSCP analysis showed that 12 out of 29 cases
of bladder cancer patients had aberrantly migrating
bands or extra bands that were further analyzed by DNA
sequencing to represent the mutations of TP53 gene
(Figure 1). All controls used in this study revealed nor-
mal migration of SSCP bands and normal sequencing of
all studied exons (Figure la,c,e).

The sequencing results confirmed that 10 cases har-
bored one or more TP53 mutations within identical or
separated samples, one case had silent mutation and one
case had bad sequencing. Eleven (37.9%) cases were clas-
sified as TP53-positive [10 cases with detected mutation

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Figure 1 Example of PCR-SSCP/HD and DNA sequencing analysis of p53 gene in transitional cell carcinoma, (a) PCR-SSCP/HD analysis of
exon 5 shows extra bands and bands with mobility shift indicated by arrows (-ve: undenaturated DNA control; +ve: denaturated DNA control,
and T: tumors). DNA sequencing reveals transition CGC^CAC at codon 175 in exon 5 (b) and insertion A after codon 244 (GGC) in exon in exon
7 (d) in compared to wild types (c) and (e) respectively (arrows).

and 1 case without DNA sequencing] and 18 (62.1%)
cases as /?53-negative [17 cases without detected muta-
tion and 1 case with silent mutation: case 5, codon 244;
GGC (Gly) to GGA (Gly)] (Table 1).

Among TP53-positive cases; seven patients (63.6%)
showed single mutation, three patients (27.3%) had
double mutations and one patient (9.1%) with mutation
detected only by SSCP analysis. A total of 13 mutations
determined in ten cases; 9 mutations (69.2%) were

single-base pair substitutions and 4 mutations (30.8%)
were with frameshift mutations.

The patterns of TP53 base-pair mutations showed that
five mutations (38.4%) were of transitions including G: C
— »A: T, three of them (23.8%) occurred at CpG di-
nucleotide in the codons (175 and 196) that were
reported as hotspot for TP53 mutations. The other two
(15.4%) mutations occurred at non-CpG sites. The four
(30.8%) transversions detected were two (15.4%) G — > C,

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Table 1 Mutational analysis of the p53 detection by SSCP and DNA sequencing, p53 nuclear reactivity and p53 status

Patients No Exon/Codon Codon change Base change Mutation type Amino acid change p53 nuclear reactivity p53 status

1

2

-

-

-

-

-

-

3

5/184

GAT^AAT

G^A

Ts

Asp184Asn

++

+

4

-

-

-

-

-

-

5

7/244

GGC^GGA

C^A

Tv

Gly244Gly

-

-

6

7/230

Del A

-

Fr

-

-

+

7
8

7/NS

-

-

-

++

+

9

-
-

-
-

-
-

-
-

-

++

-

+

10

-

-

-

-

++

+

11

-

-

-

-

-

-

12*

5/154

GGC^GGA

C^A

Tv

Gly154Gly

+

+

6/192

CAG^CAC

G^C

Tv

Glu192His

13

7/244

+ A

-

Fr

-

-

+

14

-

-

-

-

-

-

-

15

7/244

+ A

-

Fr

-

-

+

16

-

-

-

-

-

++

+

17

-

-

-

-

-

+

+

18

-

-

-

-

-

+

+

19

-

-

-

-

-

++

+

20*

6/196

CGA^CAA

G^A

Ts

Arg196Glu

++

+

8/283

CGC^CCC

G^C

Tv

Arg283Pro

21

5/175

CGC^CAC

G^A

Ts

Arg175His

++

+

22

5/176

TGC^GGC

T^G

Tv

Cys176Gly

++

+

23

++

+

24

25*

8/293

Del G

Fr

+

8/294

GAG^GAA

G^A

Ts

Glu294Glu

26

6/196

CGA^CAA

G^A

Ts

Arg196Glu

++

+

27

++

+

28

++

+

29

++

+

p53 status; + has alteration (mutation) or immunoreactivty +,++ (IHC), -; no alteration (mutation (-) and IHC (-,+/-), Ts; Transition, Tv; Transvertion, *; Tumor with
double mutation, NS; DNA not sequenced but p53 mutation detected by SSCP analysis, - G; deletion of one base (G) at codon 293, - A; deletion of one base (A) at
codon 230, + A; Insertion of one base (A) after codon 244/exon 7, Fr; Frameshift.

one (77%) T^G, and one (7.7%) C^A. Two of the
base-pair substitutions were silent mutations found in
double mutations [case 12, codon 154; GGC (Gly) to
GGA (Gly) and case 25, codon 294; GAG (Glu) to GAA
(Glu)], and seven were missense resulted in amino acid
changes.

Of the observed frameshift mutations (30.8%) three
(23.1%) were in the exon7 [two (15.4%) with insertion A
after the codon 244 (GGC/Gly) and one (7.7%) with de-
letion A at the codon 230(ACC/Thr)]. The remaining

one was deletion G (7.7%) at codon 293 (GGG/Gly) in
exon 8. The double mutations were found in case 12 in
exon 5 [codon 154; Gly — ► Gly (silent)] and in exon 6
[codon 192; Glu —> His (missense)], case 20 in exon 6
[codon 196; Arg^Glu (missense)] and exon 8 [codon
283; Arg^pro (missense)], and case 25 in exon 8
[codon 293, del G and codon 294, Glu -> Glu (silent)]
(Table 1) (Figure lb and d). The TP53 mutations were
higher in deep invasive-tumors (high grade and stage
T2) (40%) than in superficially invasive tumors (low

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grade and stage Ta-Tl) (35.7%
(Table 2).

(OR, 1.2; CI, 0.26-5.4)

Immunhistochemical analysis of TP53 gene in relation to
clinicopathological features

Immunohistochemical analysis showed that TP53 immu-
noreactivty was restricted to the nuclei of tumor cells.
Four patterns of immunohistochemical nuclear staining
was observed (Figure 2) including: no detectable immu-
noreactivty (-) in 20.7% (Figure 2,a), few focal reactivity
in less than 10% of tumor cells (+/-) in 20.7% (Figure 2,
b), heterogeneous nuclear reactivity in 10 -50% of tumor
cells (+) in 10.3% (Figure 2,c), and intense homogenous
nuclear reactivity was greater than 50% of tumor cells (+
+) in 48.3% (Figure 2,d). The level of TP53 nuclear re-
activity was classified into two categories: wild- type
TP53including score (-) and (+/-), and altered TP53
includes score (+) and (++). While, negative internal
control of samples (non-epithelial cells) showed absence
of TP53 nuclear staining that was consistent with wild-
type expression of TP53 gene (Figure 2, D). The TP53
overexpression was identified in 17 cases (58.6%) out of
29 TCC cases. The altered expression of TP53 was more
frequent in high grade tumor than low grade (66.7% vs.
28.6% respectively) and in T2 stage than Ta and Tl
(66.7% vs. 28.6% respectively), giving rise to a statistically
significant difference (p < 0.05) (Table 2).

Correlation of p53 mutation with nuclear reactivity

The present data shows no significant association be-
tween TP53 mutations and the immunohistochemical
detection of TP53 protein (p>0.05) (Table 3). Seven
tumors which had TP53 mutation [six of them with
point mutation (missense), and one case with SSCP mo-
bility shift] were positive for TP53protein overexpres-
sion. Four tumor cases with frameshift mutations of
TP53had no detectable TP53 nuclear accumulation. Ten

tumor cases without detected mutation had TP53over-
expression. Accordingly, the TP53 alteration (TP53 sta-
tus) defined by either presence of mutations or TP53
immunoreactivty, or both was demonstrated (Table 1)
[23]. Our data showed that the TP53 alteration (TP53
status) was strongly associated with high grade and stage
(p< 0.01) (Table 2).

Discussion

In Iraq, the incidence of most types of cancer (including
bladder cancer) has increased sharply in the last few
years due to exposure to wars pollution [31-33]. Muta-
tional analysis of the TP53 gene provides a unique op-
portunity to investigate the etiology, epidemiology, and
pathogenesis of human cancer [34,35]. Few studies have
been conducted on the Iraqi population following the
conflicts that led to hazardous environmental pollution.
The effects of exposure to such pollutants is hard to as-
certain as it is not possible to compare to data pre-con-
flict, which are not available either because they were
not measured or, because they were destroyed. There is
also the difficulty in finding a cohort of unexposed indi-
viduals: the conflicts were country wide and the shifting
sands spread chemical pollutants over great distances. A
definitive geographical location of where the greatest
chemical pollution exists could not be determined
[31,32], However, it is not implausible to suggest that al-
most all Iraqis have had some exposure to hazardous
and/or toxic substances. To the best of our knowledge,
the present study is the first molecular analysis of TCC
in Iraq to determine the pattern of TP53 mutations. The
results were compared with other areas polluted with
radioactive substances [15,35] and with reported specific
TP53 mutations that were known to be associated with
smoking, as smoking is a major risk factors for bladder
cancer [5].

Table 2 Association between p53 mutations as analyzed by SSCP and DNA sequencing, p53 immunoreactivty and p53
status with clinicopathological features

Variable Patients No. p53 mutation (5-8 exons) No. (%)

p53 nuclear reactivity No. (%)

p53 mut p53 wt OR 95%CI

Negative

Positive

p53 status No. (%)

P value altered Non- P value
altered

+/-

Total

Total

Total

29

1 1 (37.9)

18(62.1)

6(20.7)

6(20.7)

12(41.4)

3(10.3)

14(48.3)

1 7(58.6)

21(72.4)

8(27.6)

Grade

Low(l&[[)

14

5(35.7)

9(64.3)

4(28.6)

5(35.7)

9(64.3)

1(7.1)

4(28.6)

5(35.7)

0.02 7(50)

7(50)

1 .2 0.26-5.4

0.01

High (III)

15

6(40)

9(60)

2(13.3)

1(6.7)

3(20)

2(13.3)

10(66.7)

12(80)

14(93.3)

1(6.9)

Stage

LowO"a&T1)

14

5(35.7)

9(64.3)

4(28.6)

5(35.7)

9(64.3)

1(7.1)

4(28.6)

5(35.7)

0.02 7(50)

7(50)

1 .2 0.26-5.4

0.01

High (T2)

15

6(40)

9(60)

2(13.3)

1(6.7)

3(20)

2(13.3)

10(66.7)

12(80)

14(93.3)

1(6.9)

Negative indicates that less than 10% of tumor nuclei demonstrated p53 immunoreactivty, positive; 10% or more of tumor nuclei demonstrated p53
immunoreactivty, p53 status; + has alteration (mutation, or immunoreactivty score +,++, or both), - no alteration (mutation (-) and IHC (score -, +/-) for p53), OR,
Odds Ratio; CI, Confidence Intervals, P Value < 0.05 or 0.01 (Fisher's exact probability test).

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Figure 2 Immunohistochemical detection of p53 nuclear reactivity in transitional cell carcinoma (a) Invasive transitional cell carcinoma,
poorly differentiated (Grade III) showing no detectible nuclear p53 immunostaining (Score (-), (10X)). (b) Papillary transitional cell
carcinoma, well differentiated (Grade I) showing a few p53 immunoreactivty of tumor nuclei (Score (+/-), arrowed, (10X and 40X)). (c) Invasive
transitional cell carcinoma, poorly differentiated (Grade II) showing heterogeneous p53 nuclear immunostaining (Score (+); arrowed,(40X)). (d)
Papillary transitional cell carcinoma, well differentiated (Grade I) showing homogenous intense p53 nuclear immunostaining (Score (++); yellow
arrow, (10X and 40X), (Red arrow indicates surrounding stromal and infiltrative lymphocytes with no detectible nuclear p53 immunostaining)).

The present investigation revealed that the frequency
of TP53 mutations was higher in deep invasive-tumors
(high grade and stage T2) than superficially invasive
tumors (low grade and stage Ta-Tl) (37.9% Vs 35.7%)
(OR, 1.2; CI, 0.26-5.4) (Table 2). These observations pro-
vide further support for the proposed association be-
tween TP53 alteration and bladder cancer progression
[5,20,36]. In these results, a history of smoking was not
associated with a high frequency of TP53 mutation, nor
with the pattern of mutation in patients with TCC.

Table 3 Relation of p53 immunoreactivty to p53

mutation analyzed by SSCP and DNA sequencing

Total

p53 nuclear reactivity No. (%)
Positive Negative

P

value

p53 mutation No. (°/

3) 29

1 7(58.6)

12(41.4)

p53 mut

1 1 (37.9)

7(63.7)

4(36.3)

0.09

p53 wt

18(62.1)

10(55.6)

8(44.4)

P-value> 0.05 (Fisher's exact probability test); Mut, mutant; wt, wild-type.

Although the number of our patients is rather small,
many interesting observations are apparent. Our results
showed that the TP53 double mutations constituted
27.3% of all mutations detected, which is consistent with
values reported by Yamamoto et al, (1999) [15] who
found double mutations in their cases of dysplasia and
CIS in radio-contaminated areas. The possible explan-
ation for the occurrence of double mutations found in
our study is most likely because of the presence of a
strong carcinogenic insult from war pollution that may
have resulted in multiple transformation events [15,37].

It has been indicated that G — > A transitions were the
most prevalent type of TP53 mutation in bladder cancer;
about half of these transitions occurred at CpG sites
[38]. There is increasing evidence to indicate that CpG
bases might be more susceptible than other sites to at-
tack by environmental mutagens [39] . Studies conducted
in radio-contaminated regions in Ukraine found a high
frequency of CpG/G — > A mutations (73%) that were sig-
nificantly different from those reported by I ARC [17]. In
the present study, the frequency of all G —> A transitions

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Page 8 of 10

was 38.4% and those that occurred at CpG sites were
23.8% with predominant hotspots (2 out of 3 cases) at
codon 196. This is considered to be infrequent in blad-
der tumors [40] and may reflect the effect of certain ex-
ogenous carcinogens, such as depleted uranium, on the
frequency of mutation (Table 1).

Conversely, the transversion type of mutation that was
shown in this study (30.8%) was similar to a previously
reported finding which confirmed that TP53 mutation in
bladder cancer patients who smoked consisted of G:C —>
C:G transversions [41,42]. In our study, most TP53
mutations occurred in both smokers and non-smokers
(such as G — ► C transversions), or occurred in non-
smokers only (such as C —> A and T — > G transversions).
This observation unveils new evidence for the presence
of risk factors for cancer, other than cigarette smoking,
that may underlie this type of mutation.

Another important observation was the frameshift
mutations which were found to be higher (30.8%) in fre-
quency than what has been reported previously [5,43],
and was found preferentially to occur in exon-7 . Two
the frameshift mutation having the same mutation (in-
sertion A after codon 244) that cannot be excluded as a
relative hotspot (Table 1). In fact, the most common
mutations observed in TP53 DNA binding (exons 5-8)
were missense mutations while frameshift types were
found to be less frequent [43]. Most studies concerning
the mutational signature of p53 in relation to the history
of smoking revealed that the frameshift mutation in the
DNA binding either have not been observed [44] or oc-
curred less frequently than point mutation (7% and
7.1%) with different types of deletions and insertions
[7,40]. In radio-contaminated regions in Ukraine, all
TP53 mutations determined were single-bp substitu-
tions; no base deletions or insertions were found [15,45].
This indicated a possible distinct molecular carcinogen-
esis pathway for bladder cancer after the Chernobyl dis-
aster, based on a different incidence of p53 gene
mutations compared with tumors found in the same
population before the accident [45].

Accordingly, frameshift mutations (especially insertion
A) of TP53 may reflect the effect of certain exogenous
environmental contamination and may be considered as
a useful predictor marker for bladder cancer. No reports
on the levels of environmental pollution from toxic che-
micals or radiation exist as no studies have been con-
ducted to measure and specify these effects with
accuracy. This study and future studies on the health of
the Iraqi populace are needed to stimulate investigations
on the extent and localization of environmental pollu-
tion caused by the recent conflicts.

In fact, the occurrence of TP53mutations leads to con-
formational changes of the protein, resulting in a pro-
longed half-life and subsequent accumulation of mutations

in the nuclei. The extended half-life of the protein is the
basis for immunohistochemical detection of TP53
[20,22,44]. Whilst immunohistochemical positivity for
TP53 protein generally reflects point mutations of TP53
genes in tumor cells, it is not always synonymous with
mutations [20,22]. The present study revealed that the
TP53 overexpression (58.6%) was observed more fre-
quently in high grade and in T2 stage than in low grade
and Ta and Tl tumors (p < 0.05) (Table 2). This is consist-
ent with other findings which reported that the TP53 over-
expression was associated with high grade and stage of
bladder cancer [20,46] .

It has been reported that there is a good concordance
between overexpression and mutation of TP53 gene
[20,22], however, other studies have shown a consider-
able discrepancy between them [47,48]. Analysis of the
data in this study showed no significant association be-
tween TP53 mutations and the immunohistochemical
detection of TP53 protein (p>0.05) (Table 3). This is
largely because of the four tumors with frameshift muta-
tions that had no detectable TP53 protein. These muta-
tions encoded deleted or truncated proteins that are
very unstable in the cell and usually not detectable by
IHC method even when using an antibody containing
the corresponding N-terminal epitope [49,50]. The
tumors with missense mutations were positive for TP53
protein overexpression (Table 3); missense mutations
resulting in amino acid change render the TP53 protein
a more stable compound with a longer half-life that can
be detected by standard immunhistochemical methods
[20,44].

On the other hand, the presence of other tumors that
demonstrated TP53 overexpression without mutations
might be due to mutations in the TP53 that occurred
outside exons 5-8 [49], or to the overexpression of
TP53 caused not only by TP53 mutations but also by
other factors (such as MDM2) which bind to TP53 pro-
tein, thus increasing its half-life and allowing it to accu-
mulate in the nucleus. This observation may reflect
alterations in the TP53 pathway rather than in the TP53
gene itself [6,51]. However, the antibody used in our
study recognises both the wild-type and the mutant
forms of TP53 protein at the same time. Hence, it is
possible that the IHC assay detects accumulation of the
wild-type in some cases. The level of wild-type TP53
protein can be increased in response to DNA damage,
hypoxia, oncogene activation and changes in the nucleo-
tide pool, which are commonly observed in primary
tumors [51,52]. Furthermore, up-regulation of wild-type
TP53 protein in tumors may indicate the last step of de-
fence before metastasis [53]. Therefore, the TP53 alter-
ation (TP53status) that is used as a prognostic marker of
bladder tumorigenesis should be determined by combin-
ation of mutational and IHC analysis. Accordingly, the

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Page 9 of 10

present investigation of TP53 status has shown a strong
association with deep invasive tumors (p< 0.01) (Table 2).
The altered expression of TP53 tumor suppressor gene
is an independent predictor of bladder cancer progres-
sion when examined as an individual determinant
[10,20].

Conclusions

The TP53 frameshift mutations (especially insertion A)
and 196 hotspot codon may represent a possible specific-
mutational patterns associated with bladder tumorigen-
esis, and reflect a preferential target for exogenous carci-
nogens. Furthermore, the difference in incidence and type
of TP53 mutations among the Iraqi TCC patients may ex-
plicitly indicate a distinct molecular pathway responsible
for the development of bladder cancer due to exposure to
environmental hazards (e.g. depleted uranium). The un-
usual mutation patterns of TP53 necessitates a complete
molecular epidemiological study for further clarification of
distinct molecular pathways for bladder cancer pathogen-
esis among Iraqi patients. The current study confirmed
that the combination of molecular analysis and protein ex-
pression of TP53 tumor suppressor gene is highly recom-
mended for studying gene alterations in bladder cancer
rather than the application of a single approach.

Abbreviations

LSAB+: Labeled Streptavidin-biotin; BC: Bladder Cancer; TCC: Transitional Cell
Carcinoma; FFPE: Formalin-Fixed Paraffin-Embedded Tissue; DU: Depleted
Uranium; IHC: Immunohistochemistry; PCR: Polymerase chain reaction;
TUR: Transurethral; SSCP: Single strand conformation polymorphism.

Competing interests

The authors declare that they have no competing interests.
Authors' contributions

ThAA and MH carried out the molecular genetic studies. THAA performed
the statistical analysis. AA and AKM initiated the project at the University of
Kufa . DA and AA carried out the immunohistochemical analysis and
histopathological examination. MR participated in the molecular genetic
studies. MNA provided the surgical specimens. AAY and AA conceived of the
study, participated in its design and drafted the manuscript. All authors read
and approved the final manuscript.

Author details

1 Middle Euphrates unit for cancer research, Faculty of Medicine, University of
Kufa, Kufa, Iraq. 2 National Institute for Genetic Engineering and
Biotechnology, Tehran, Iran, department of Pathology and Forensic
Medicine, Faculty of Medicine, University of Kufa, Kufa, Iraq, biotechnology
Department, College of Science, Baghdad university, Baghdad, Iraq. 5 Urology
department, Faculty of Medicine, University of Kufa, Kufa, Iraq, department
of Pathology and Forensic Medicine, Faculty of Medicine, University of Kufa,
Kufa, P.O. Box 21, Najaf Governorate, Iraq.

Received: 3 June 2012 Accepted: 17 August 2012
Published: 28 August 2012

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